Fusion protein having enhanced in vivo activity of erythropoietin

ABSTRACT

The present invention relates to a fusion protein having enhanced in vivo activity of erythropoietin wherein a carboxy terminal peptide fragment of thrombopoietin is fused with the carboxy terminal of human erythropoietin. This fusion protein has highly enhanced in vivo half-life due to increased carbohydrate content without loss of the inherent activity of erythropoietin, and does not cause any antigenicity when applied to the human body.

This application claims the prior benefit of Korean Patent ApplicationNo. 2001-74975, filed on Nov. 29, 2001.

TECHNICAL FIELD

The present invention relates to a fusion protein having enhanced invivo activity of erythropoietin (EPO, below) that is a new medicine forthe treatment of anemia. Specifically, the present invention relates toa fusion protein having highly enhanced in vivo half-life and activityof erythropoietin by fusion of EPO molecule with a certain peptide thathas half-life elongation activity and is derived from the human body.

BACKGROUND ART

EPO, a glycoprotein having the molecular weight of 30,000 to 34,000, isa factor that stimulates production and differentiation of red bloodcells. This protein acts by binding to receptors on erythrocyteprecursor cells to result in increase of calcium ion concentration in acell, increase of DNA biosynthesis, and stimulation for the formation ofhemoglobin and the like. This EPO can be used for the treatment ofanemia from renal failure, anemia of a premature baby, anemia fromhypothyroidism, anemia from malnutrition, etc. The clinical use ofrecombinant human EPO is on the increase. However, such use may causesome inconvenience and high costs because it should be administered onthe average three times a week due to its short half-life. Thus, if thein vivo activity of EPO is maintained for a long time, theadministration frequency of EPO may be greatly decreased.

Efficacy of EPO is proportional to in vivo half-life thereof. It isknown that in vivo half-life of EPO is correlated to the content ofsialic acid that is located at the terminal of carbohydrate chains ofEPO. Therefore, efficacy of EPO is highly dependent on the presence ofcarbohydrate chains. Since the forms of carbohydrates appear differentlydepending on the kind of cells where EPO is expressed, the sameglycoproteins may have different carbohydrate structure if they areexpressed in different cells. Although it has been recently demonstratedthat some bacteria can attach the carbohydrate chains, typical bacteria,for example E. coli, are known not to do. Proteins expressed in E. colido not contain the carbohydrate chains, and thus, E. coli-derived EPO,which does not contain the carbohydrate chains, exhibits positive invitro activity but no in vivo activity. It is because deglycosylated EPOis rapidly eliminated from the human body and has extremely shorthalf-life. In conclusion, the carbohydrate chains play a very importantrole in the activity of EPO.

Many studies have been made to enhance the activity of EPO. The mainapproach is substitutions of some amino acids of EPO by mutagenesis onthe EPO gene. For example, PCT/US94/09257, filed by Amgen and titled“Erythropoietin analogs,” discloses a method to increase in vivohalf-life of EPO by increasing the carbohydrate contents throughmutagenesis. Also, an attempt to increase in vivo half-life of EPO wasmade by formation of EPO dimer. See, A. J. Sytkowski et al., J.B.C. vol.274, No. 35, pp24773–24778. Besides, another known method is to enhancein vivo activity of EPO by fusing new amino acids, peptides, or proteinfragments with EPO molecule in the genetic engineering manner andincreasing the carbohydrate content, i.e., sialic acid content of EPO.However, all amino acids, peptides, or heterogeneous protein fragmentsmay not be used for this purpose. In most cases, such modifications mayresult in decrease or loss of inherent activity of protein and may causea problem of antigenicity when used in vivo.

Although it is not related to EPO, fusion proteins or chimeric proteinshave been studied, for example, for follicle stimulating hormone that isa kind of sex hormone. See, Furuhashi et al., 1995, Mol. Endocrinol.However, the methods have not been applied to the industry since proteinmodifications using a genetic engineering method have many risks. Thatis, in most cases, the target protein may not be readily obtainedwithout professional skills, and on the contrary, the inherent activityof protein may be decreased or lost by the addition or substitution ofnew amino acids.

DISCLOSURE OF THE INVENTION

The present inventors have extensively studied new methods for enhancingin vivo activity of EPO by fusing new amino acids, peptides, or proteinfragments with EPO molecule, and so increasing the carbohydrate contentthereof. As a result, the present inventors have discovered that afusion protein of EPO obtained by fusing a carboxy terminal peptide(CTP, below) of thrombopoietin (TPO, below), a protein that alreadyexists in the human body, with the carboxy terminal of EPO has highlyenhanced in vivo half-life due to a lot of amino acids that increaseglycosylation site without loss of the inherent activity of EPO, anddoes not cause any antigenicity when applied to the human body. Then,the present inventors have completed the present invention.

Therefore, the object of the present invention is to provide a fusionprotein having enhanced in vivo activity of human EPO and containing CTPof TPO fused with human EPO at the carboxy terminal thereof.

Another object of the present invention is to provide a nucleic acidencoding the fusion protein, a recombinant vector containing the nucleicacid, and a host cell line transformed with the recombinant vector.

Further object of the present invention is to provide a process forpreparation of the fusion protein having enhanced in vivo activity ofhuman EPO by cultivating the transformed cell line.

First, the present invention relates to a fusion protein having enhancedin vivo activity of human EPO and containing CTP of TPO fused with humanEPO at the carboxy terminal thereof. The CTP preferably comprises theamino acid sequence of SEQ ID No. 1 that corresponds to amino acids ofpositions 176 to 353 (LTP, below) or parts thereof, particularly theamino acid sequence of SEQ ID No. 2 that corresponds to amino acids ofpositions 337 to 353 (STP, below).

Particularly, the fusion protein according to the present invention maycomprise the amino acid sequence of SEQ ID No. 3 or 4.

Second, the present invention relates to a nucleic acid encoding thefusion protein, a recombinant vector containing the nucleic acid, and ahost cell line, preferably CHO (Chinese hamster ovary) cell, transformedwith the recombinant vector.

Third, the present invention relates to a process for preparation of thefusion protein having enhanced in vivo activity of human EPO bycultivating the transformed cell line.

Below, the present invention will be explained in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a and 1 b show the nucleotides (SEQ ID NOS 15 & 16) and aminoacid sequences (SEQ ID NOS 1 & 2) of carboxy terminal peptides (LTP,STP) of TPO;

FIGS. 2 aa and 2 ab show the nucleotide (SEQ ID NO: 17) and amino acidsequence (SEQ ID NO: 3) of ELTP that is a fusion protein of EPO and thepeptide LTP, and FIG. 2 b shows the nucleotide (SEQ ID NO: 18) and aminoacid sequence (SEQ ID NO: 4) of ESTP that is a fusion protein of EPO andthe peptide STP;

FIGS. 3 a and 3 b are schemes showing the procedures to prepare theexpression vectors, pcDNA-ELTP and pcDNA-ESTP;

FIG. 4 is an electrophoresis photograph of expressed ELTP and ESTP; and

FIG. 5 is a graph showing the pharmacokinetic results of EPO, ELTP, andESTP.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention comprises the steps of preparation and cloning ofa gene of the desired fusion protein, construction of an expressionvector containing the desired gene, transformation of an animal cell,expression, purification, and biological assay.

(1) Preparation of Gene

EPO cDNA may be obtained by performing the conventional PCR technique(PCR PreMix Kit of Bioneer Co.) using the complementary primers of EPOcDNA terminals (EP1 and EP2) from cDNA library of human-derived fetalliver (Invitrogen Co.). TPO cDNA may be obtained using the complementaryprimers of TPO cDNA terminals (T1 and T2) according to the same manner.

EP1: ATGGGGGCACGAATGTCCTGCCTGGCTGG (SEQ ID No.5) EP2:GTCCCCTGTCCTGCAGGCCT (SEQ ID No.6) T1: ATGGATCTGACTGAATTGCTCCTC (SEQ IDNo.7) T2: TTACCCTTCCTGAGACAGATTCTGGGA (SEQ ID No.8)

EPO cDNA and TPO cDNA obtained by PCR are cloned to pGEM-T (PromegaCo.), a cloning vector, respectively. The pGEM-EPO and pGEM-TPO are thensequenced, and used as a template for the following procedures.

LTP, a CTP gene of TPO used in the present invention, may be obtained byperforming PCR using pGEM-TPO as a template and primers EL1 and T2.

EL1: GAGGCCTGCAGGACAGGGGACGCCCCACCCACCACAGCTGTCC (SEQ ID No. 9)

The primer EL1 contains a gene extended to the position of restrictionenzyme StuI, which is located near the 3′ terminal of EPO, and a part of5′ terminal of LTP gene (amino acids of positions 176 to 353 of TPO) atthe same time. On the other hand, PCR is performed using pGEM-EPO as atemplate and primers EP1 and EP2 to give EPO gene only. Thus obtainedEPO and LTP genes are treated with restriction enzyme StuI,respectively. Then, fragments of EPO and LTP genes are ligated and theligation product is used as a template in the PCR procedure usingprimers EP 11 and L2 to give a gene fragment ELTP having about 1113 bps.

EP11: TAAGCTTATGGGGGTGCACGAATGT (SEQ ID No.10) L2:TGGATTCTTACCCTTCCTGAGACAGATTC (SEQ ID No.11)

This gene is cloned to a cloning vector of pGEM-T and then sequenced(pGEM-ELTP, FIG. 3 a).

Further, the STP gene used in the present invention may be obtained bysynthesis and self-priming PCR procedure. Synthesized gene fragments arethe following ES1, S2, S3 and the above L2.

ES1: AGGGGAGGCCTGCAGGACAGGGGACCCTCTTCTAA (SEQ ID No.12) S2:GTGGGTGTAGGATGTGTTTAGAAGAGG (SEQ ID No.13) S3: TACACCCACTCCCAGAATCTGTCTC(SEQ ID No.14)

1 μl (50 pmole/μl) of each 4 gene fragments is taken and PCR isperformed using high fidelity Taq system of BM Co.

The gene fragment of about 50 bps (STP gene) is identified in 1% agarosegel. This gene encodes the 17 amino acids (amino acids of positions 337to 353) of the carboxy terminal of TPO (FIG. 1).

PCR is performed using pGEM-EPO as a template and primers EP11 and EP2to give EPO gene only. This EPO gene and the STP gene are used astemplates at the same time and primers EP11 and L2 are used in a PCRusing the high fidelity Taq system of BM Co. to give the desired fusiongene, ESTP gene, of about 630 bps. This gene is cloned to pGEM-T, acloning vector, and then sequenced (pGEM-ESTP, FIG. 3 b).

(2) Construction of Expression Vector

pcDNA3.1 vector of Invitrogen Co. is used as an expression vector.

The pcDNA3.1 vector is treated with restriction enzymes HindIII andBamHI to make a linear vector, and PGEM-ELTP and pGEM-ESTP are alsotreated with the same restriction enzymes, HindIII and BamHI,respectively. The digested pcDNA3.1, ELTP, and ESTP genes are isolatedusing Qiagen extraction kit on agarose gel. After each ligation of thegenes, the ligation product is introduced into E. coli NM522. Plasmidsare isolated from the colonies that have been formed from cultivation ofthe transformed E. coli in LB-ampicillin plate overnight, and aretreated with restriction enzymes, HindIII and BamHI. Colonies havingELTP or ESTP gene are then screened by 1% agarose gel electrophoresis.These plasmids are designated as pcDNA-ELTP and pcDNA-ESTP, respectively(FIGS. 3 a and 3 b).

(3) Transformation of CHO Cell and Expression

CHO cells (DG44) are cultivated to a confluency of 40˜80% (1˜4×10⁵cells/60 mm dish) in a 60 mm cell culture dish. 3 μl of superfectionreagent (BM Co.) and 97 μl of cell culture medium (α-MEM with media, noserum, no antibiotics) are mixed well, and pcDNA-ELTP (=0.1 μg/μl, about2 μg) and vector pLTRdhfr26 (ATCC37295, 0.2 μg) having dhfr gene areadded thereto. The mixture is reacted for 5˜10 minutes at roomtemperature and then added to the cells as prepared above. After oneday, the medium is refreshed with a medium containing G418 in an amountof 500 μg/ml (α-MEM without media, 10% FBS). The medium is thenrefreshed with the G418 medium containing 500 μg/ml of G418 for 7˜10days, during which cells without G418 resistance gene and negativecontrol cells are completely killed. Selected cells on G418 medium arecultivated sufficiently and an expressed ELTP protein is identifiedusing EPO ELISA kit of BM Co. The same process is applied to ESTPexpression vector and then an expressed ESTP fusion protein is alsoidentified in the same manner.

(4) Purification of the Expressed ELTP and ESTP

Affinity resin for isolation and purification is prepared using anti-EPOmonoclonal antibody (R&D Co.) as follows:

0.3 g of CNBr-activated Sepharose 4B is swelled for 20 minutes in 1 mMHCl, and the resin is moved to a column and washed with 1 mM HCl. Theresin is washed with 4 ml of coupling buffer (0.1M NaHCO₃, 0.5M NaCl, pH8.3), immediately mixed with anti-EPO monoclonal antibody (500 μg/vial)contained in 4 ml of coupling buffer, and reacted for 2 hours at roomtemperature with stirring the tube. Refreshed with blocking buffer (0.2Mglycine, pH 8.0), the resin is reacted at room temperature for 2 hourswith stirring the tube. Then, the resin is washed with 6.5 ml ofcoupling buffer, 6.5 ml of acetate buffer (0.1M acetic acid, 0.5M NaCl,pH 4), and 6.5 ml of coupling buffer in the order. A column is preparedusing such obtained resin and purification is conducted as describedbelow.

Cells are cultivated in serum free medium for one day, and the medium isconcentrated to about 5 folds using Centriprep (Millipore MWCO 10,000).This concentrate is loaded to a column equilibrated in advance with PBSat a flow rate of 20 ml/hr, and washed again with PBS. Elution buffer(0.1 M glycine, pH 2.8) is applied to the column and the eluent isimmediately titrated with 1M Tris to pH 7.5. The purity is at least 97%when analyzed by SDS-PAGE and silver staining (FIG. 4).

(5) Activity Measurement by Bioassay Method

In the bioassay test using mouse spleen cells treated withphenylhydrazine, the ELTP and ESTP, which are expressed and properlypurified, show higher activity than EPO. It demonstrates that the fusedcarboxy terminals in the ELTP and ESTP may not inhibit activity of EPOitself.

(6) Pharmacokinetic Experiments

Pharmacokinetic experiments are performed to mice to determine whetherthe prepared ELTP and ESTP actually have prolonged in vivo half-life.The candidate substance is intravenously administered to 4 mice in anamount of 20 units/mouse. In order to identify the time-lapseconcentration in blood, blood is taken from the mice at an interval of30 minutes and concentrations are measured using EIA kit of BoehringerMannheim Co. In the pharmacokinetic experiments to mice, the candidatesubstances, ELTP and ESTP, show far longer half-life than the controlEPO (FIG. 5).

The present invention will be more specifically explained in thefollowing examples. However, it should be understood that the followingexamples are intended to illustrate the present invention but not tolimit the scope of the present invention in any manner.

EXAMPLE 1 Preparation of Gene

EPO cDNA was obtained by performing the conventional PCR (PCR PreMix Kitof Bioneer Co.) using the primers EP1 and EP2 that are complementary tothe terminals of EPO cDNA, in an amount of 50 pmole, respectively, fromcDNA library of human-derived fetal liver (Invitrogen Co.). TPO cDNA wasobtained using the primers T1 and T2, complementary to the terminals ofTPO cDNA, according to the same manner. Total 30 cycles of PCR wereperformed using high fidelity Taq system of BM Co. under the conditionof 52° C. for 40 seconds for annealing, 72° C. for 55 seconds, and 94°C. for 20 seconds to give EPO cDNA and TPO cDNA. They were cloned topGEM-T (Promega Co.), a cloning vector, respectively. That is, the PCRproducts were eluted from 1% agarose gel and ligated into pGEM-T, whichwas then introduced into E. coli NM522. The transformed E. coli wascultivated overnight in LB-ampicillin plate containing X-gal/IPTG.Plasmids were purified from the white colonies and treated withrestriction enzymes, SacI and SacII, to screen the colonies containingthe respective cDNAs. The vectors obtained at this stage were designatedas pGEM-EPO and pGEM-TPO, which were then sequenced, and used astemplates for the following procedures.

LTP, CTP gene of TPO used in the present invention, was obtained byperforming total 30 cycles of PCR using the high fidelity Taq system ofBM Co. and using pGEM-TPO as a template and primers, EL1 and T2 (50pmole), under the condition of 50° C. for 40 seconds for annealing, 72°C. for 45 seconds, and 94° C. for 20 seconds. The primer EL1 contains agene from the position of restriction enzyme StuI, which is located nearthe 3′ terminal of EPO, to the 3′ terminal, and a part of 5′ terminal ofLTP gene at the same time. A gene fragment of about 534 bps wasidentified on 1% agarose gel (LTP gene). This gene encodes the 178 aminoacids of the carboxy terminal of TPO (amino acids of positions 176 to353) (FIG. 1).

On the other hand, PCR was performed using pGEM-EPO as a template andprimers EP1 and EP2 to give EPO gene only.

Thus obtained EPO and LTP genes were treated with restriction enzymeStuI, respectively. Then, fragments of EPO and LTP genes were purifiedby Qiagen extraction kit and ligated with a ligase. The ligation productof EPO and LTP genes was used as a template in total 30 cycles of PCRprocedure using primers EP11 and L2 (50 pmole) and using the highfidelity Taq system of BM Co. under the condition of 55° C. for 40seconds for annealing, 72° C. for 60 seconds, and 94° C. for 20 secondsto give the desired fusion gene of ELTP having about 1113 bps. This genewas cloned to a cloning vector of pGEM-T in the same manner as above andthen sequenced (pGEM-ELTP, FIG. 3 a).

STP gene was obtained by synthesis and self-priming PCR procedure.Synthesized DNA fragments were ES1, S2, S3 and L2. 1 μl (50 pmole/μl) ofeach of the 4 DNA fragments was taken and total 15 cycles of PCR wereperformed using the high fidelity Taq system of BM Co. under thecondition of 55° C. for 40 seconds for annealing, 72° C. for 40 seconds,and 94° C. for 20 seconds. A gene fragment of about 50 bps wasidentified on 1% agarose gel (STP gene). This gene encodes the 17 aminoacids of the carboxy terminal of TPO (amino acids of positions 337 to353) (FIG. 1).

PCR was performed using pGEM-EPO as a template and primers EP11 and EP2in the same manner as above to give EPO gene only. This EPO gene and theSTP gene were used as templates at the same time and primers of EP11 andL2 were used in total 30 cycles of PCR using high fidelity Taq system ofBM Co. under the condition of 58° C. for 40 seconds for annealing, 72°C. for 50 seconds, and 94° C. for 20 seconds to give the desired fusiongene, ESTP gene, of about 630 bps. This gene was cloned to pGEM-T, acloning vector, and then sequenced (pGEM-ESTP, FIG. 3 b).

EXAMPLE 2 Construction of Expression Vectors pcDNA-ELTP and pcDNA-ESTP

pcDNA3.1 vector of Invitrogen Co. was used as an expression vector. ELTPand ESTP genes that are cloned to pGEM-T vector contain HindIII andBamHI recognition sites at terminals, respectively. pcDNA3.1, pGEM-ELTPand pGEM-ESTP were treated with restriction enzymes, HindIII and BamHI,respectively. The linearized pcDNA3.1, ELTP, and ESTP genes wereisolated using Qiagen extraction kit on agarose gel. After ligation ofpcDNA3.1 with ELTP or ESTP, the ligation product was introduced into E.coli NM522. Plasmids were isolated from the colonies that had beenformed from cultivation of the transformed E. coli in LB-ampicillinplate overnight, and were treated with restriction enzymes, HindIII andBamHI. Colonies having ELTP or ESTP gene were then screened by 1%agarose gel electrophoresis. These plasmids were designated aspcDNA-ELTP and pcDNA-ESTP, respectively (FIG. 3).

EXAMPLE 3 Transformation of CHO Cell and Expression

CHO cells (DG44) were cultivated to a confluency of 40˜80% (1˜4×10⁵cells/60 mm dish) in a 60 mm cell culture dish. 3 μl of superfectionreagent (BM Co.) and 97 μl of cell culture medium (α-MEM with media, noserum, no antibiotics) were mixed well, and plasmid pcDNA-ELTP (=0.1μg/μl, about 2 μg) and vector pLTRdhfr26 (ATCC37295, 0.2 μg) having dhfrgene were added thereto. The mixture was reacted for 5˜10 minutes atroom temperature and then added to the cells as prepared above. Afterone day, the medium was refreshed with a medium containing G418 in anamount of 500 μg/ml (α-MEM without media, 10% FBS). The medium was thenrefreshed with the G418 medium containing 500 μg/ml of G418 for 7˜10days, during which cells without G418 resistance gene and negativecontrol cells were completely killed. Selected cells on G418 medium werecultivated sufficiently and an expressed ELTP protein was identifiedusing EPO ELISA kit of BM Co. The same process was applied topcDNA-ESTP.

EXAMPLE 4 Purification of the Expressed Fusion Proteins

Affinity resin for isolation and purification was prepared usinganti-EPO monoclonal antibody (R&D Systems Co.) as follows:

0.3 g of CNBr-activated Sepharose 4B was swelled for 20 minutes in 1 mMHCl, and the resin was moved to a column and washed with 1 mM HCl. Theresin was washed with 4 ml of coupling buffer (0.1M NaHCO₃, 0.5M NaCl,pH 8.3), immediately mixed with anti-EPO monoclonal antibody (500μg/vial) contained in 4 ml of coupling buffer in a tube, and reacted for2 hours at room temperature with stirring the tube. Refreshed withblocking buffer (0.2M glycine, pH 8.0), the resin was reacted at roomtemperature for 2 hours with stirring the tube. Then, the resin waswashed with 6.5 ml of coupling buffer, 6.5 ml of acetate buffer (0.1Macetic acid, 0.5M NaCl, pH 4), and 6.5 ml of coupling buffer in theorder. A column was prepared using such obtained resin and purificationwas conducted as described below.

Cells were cultivated in serum free medium for one day, and the mediumwas concentrated to about 5 folds using Centriprep (Millipore, MWCO10,000). This concentrate was loaded to a column equilibrated in advancewith PBS at a flow rate of 20 ml/hr, and washed again with PBS. Elutionbuffer (0.1M glycine, pH 2.8) was applied to the column and the eluentwas immediately titrated with 1M Tris to pH 7.5. SDS-PAGE results of thepurified ELTP and ESTP are shown in FIG. 4. The purity was at least 97%when analyzed by SDS-PAGE and silver staining.

EXAMPLE 5 Activity Measurement by Bioassay Method

Phenylhydrazine was injected to a mouse once a day for 2 days at a doseof 60 mg/kg. After 3 days, enlarged spleen was separated and ground witha homogenizer to obtain spleen cells. The spleen cells were diluted to6×10⁶ cells/ml and 100 μl of the cell solution was introduced into eachwell of a 96 well plate. 0˜500 mU/ml of authentic EPO and 100 mU/ml ofthe expressed fusion protein were added to each well, and the plate wasallowed to stand at 37° C. for 22 hours in a CO₂ incubator. 50 μl ofdimethyl ³[H]-thymidine (20 μCi/ml) was added to each well, furtherreacted for 2 hours in the same incubator, and the reaction solution wasthen adsorbed onto a glass filter (Nunc 1–73164) per each well. Thefilters were washed with physiological saline 3 times and the amount ofradioactivity of each filter was measured using β counter. Theactivities of the fusion proteins, ELTP and ESTP were shown to becomparable to or higher than that of authentic EPO, which demonstratedthat the carboxy terminals fused into EPO did not inhibit activity ofEPO itself.

EXAMPLE 6 Pharmacokinetic Experiments

Pharmacokinetic experiments were performed to mice to determine whetherthe prepared candidate substance actually has prolonged in vivohalf-life. The fusion protein purified according to the process ofExample 5 was intravenously administered to 4 mice in an amount of 20units/mouse. In order to identify the time-lapse concentration in blood,blood was taken from the mice at an interval of 30 minutes andconcentrations were measured using EIA kit of Boehringer Mannheim Co.The results are shown in FIG. 5. As can be seen from FIG. 5, ELTP andESTP show about three times longer half-life than the control, EPO,respectively (half-life of EPO was 22 minutes, that of ELTP was 60minutes, and that of ESTP was 57 minutes).

INDUSTRIAL APPLICABILITY

The fusion proteins according to the present invention have highlyincreased in vivo half-life of EPO due to the presence of amino acidsthat increase the carbohydrate chains without influencing the inherentactivity of EPO. Further, the fusion proteins do not cause any problemof antigenicity when applied to the human body since the fused peptide,LTP or STP, is a peptide that already exists in the body.

1. A fusion protein wherein a carboxy terminal peptide (CTP) ofthrombopoietin (TPO) consisting of the amino acid sequence of SEQ ID NO.2 is fused with human erythropoietin (EPO) at the carboxy terminal ofthe human EPO.
 2. A nucleic acid encoding the fusion protein accordingto claim
 1. 3. A process for preparation of a fusion protein havingenhanced in vivo activity of human EPO-compared to wild type human EPOcomprising cultivation of a host cell line transformed with arecombinant vector containing the nucleic acid according to claim 2.